Background/Objectives: Tissue factor (TF)-expressing cancer cells and their extracellular vesicles (CaCe-dEVs) are key drivers of cancer-associated hypercoagulability and vascular dysfunction. While low-molecular-weight heparins (LMWHs) and direct FXa inhibitors are standard therapies for cancer-associated thrombosis, their direct effects on cancer cell procoagulant potential and endothelial responses remain incompletely defined. This study compared the impact of LMWHs (enoxaparin, tinzaparin), apixaban, and quercetin on cancer cell viability, thrombin generation, and CaCe-dEVs-induced endothelial injury. Methods: Pancreatic (BXPC3) and breast (MCF7) cancer cells and their vesicles were analyzed for TF expression and thrombin generation. Human umbilical vein endothelial cells (HUVECs) were pretreated with each agent prior to vesicle exposure. Cell viability, thrombin generation, and endothelial morphology were assessed using standard assays and microscopy. Results: Tinzaparin and quercetin significantly reduced cancer cell viability, whereas enoxaparin and apixaban showed no cytotoxicity. None of the agents affected HUVEC viability. All suppressed TF-mediated thrombin generation induced by cancer cells, with tinzaparin being most effective in BXPC3 cells. Quercetin exhibited a partial and limited protective effect on endothelial cells against CaCe-dEVs-induced dysfunction, while LMWHs and apixaban did not prevent endothelial damage. Conclusions: These findings suggest that LMWHs, apixaban, and quercetin modulate cancer-cell-driven hypercoagulability beyond anticoagulation, with quercetin and tinzaparin showing additional cytotoxic potential. Such dual effects may reduce thrombosis risk while impacting tumor progression, meriting further investigation.

Background/Objectives: Tissue factor (TF)-expressing cancer cells and their extracellular vesicles (CaCe-dEVs) are key drivers of cancer-associated hypercoagulability and vascular dysfunction. While low-molecular-weight heparins (LMWHs) and direct FXa inhibitors are standard therapies for cancer-associated thrombosis, their direct effects on cancer cell procoagulant potential and endothelial responses remain incompletely defined. This study compared the impact of LMWHs (enoxaparin, tinzaparin), apixaban, and quercetin on cancer cell viability, thrombin generation, and CaCe-dEVs–induced endothelial injury. Methods: Pancreatic (BXPC3) and breast (MCF7) cancer cells and their vesicles were analyzed for TF expression and thrombin generation. Human umbilical vein endothelial cells (HUVEC) were pretreated with each agent prior to vesicle exposure. Cell viability, thrombin generation, and endothelial morphology were assessed using standard assays and microscopy. Results: Tinzaparin and quercetin significantly reduced cancer cell viability, whereas enoxaparin and apixaban showed no cytotoxicity. None of the agents affected HUVEC viability. All suppressed TF-mediated thrombin generation induced by cancer cells, with tinzaparin being most effective in BXPC3 cells. Quercetin consistently protected endothelial cells from CaCe-dEVs–induced dysfunction, while LMWHs and apixaban did not prevent endothelial damage. Conclusions: These findings suggest that LMWHs, apixaban, and quercetin modulate cancer cell-driven hypercoagulability beyond anticoagulation, with quercetin and tinzaparin showing additional cytotoxic potential. Such dual effects may reduce thrombosis risk while impacting tumor progression, meriting further investigation.

Background: Thromboinflammation is recognized as a central pathological process in sepsis. Sepsis-induced inflammation, triggered by bacterial infection, is tightly linked to a hypercoagulable state and endothelial cell activation. The failure of natural anticoagulant pathways—due to overwhelming thromboinflammation and/or consumption of natural coagulation inhibitors—contributes to the development of disseminated intravascular coagulation (DIC) and clinical deterioration. Excessive expression of tissue factor (TF) by activated endothelial cells and monocytes plays a key role in this process. Release of procoagulant microparticles (MP) is related with amplification of blood borne hypercoagulability in patients with sepsis. The identification of septic patients at risk of deterioration aiming to prompt administration of antithrombotic treatment remains an unmet need. To this aim, the identification of new biomarkers of hypercoagulability is a promising strategy. Among emerging candidates, TF-bearing microparticles (TF⁺-MPs) represent a particularly compelling biomarker with both mechanistic and clinical relevance. Aim: The ROADMAP-Sepsis study evaluated the clinical relevance of TF⁺ -MP activity quantification in relation to systemic inflammation, hypercoagulability, endothelial cell dysfunction and 20-day mortality in patients with sepsis, stratified by DIC status. Methods: In this prospective, observational study, 114 ICU patients with sepsis (according to the Sepsis-3 criteria: documented infection and SOFA score ≥2) and without prior antithrombotic treatment were enrolled. Patients were stratified according to the ISTH-DIC score into a DIC (score ≥5; n=65) and non-DIC groups (n=49). Blood samples were collected within 2 hours of ICU admission into citrated tubes (Vacutainer®). Platelet-poor plasma (PPP) was prepared using standardized protocols for MP analysis. TF⁺-MP activity was measured using a TF-dependent FXa generation assay (CY-Quant MV-TF Activity, Biocytex, France), and total MP concentration was assessed with the ZYMUPHEN MP kit (Hyphen Biomed, France). D-dimer, fibrin degradation products (FDPs), soluble thrombomodulin (sTM), tissue factor pathway inhibitor (TFPI), CRP, IL-1β , Procalcitonin (PCT), and TNF-α , were measured with ELISA kits (Diagnostica Stago). Thrombin generation (TG) in PPP was analyzed by calibrated automated thrombogram (CAT®, Stago) with 5 pM TF PPP-Reagent®. The primary clinical endpoint was 20-day mortality. Results: The DIC group had a significantly higher ISTH score compared to the non-DIC group (p < 0.001). Total MP concentrations did not differ significantly between groups and showed no correlation with DIC or SOFA scores, nor with D-dimer, FDPs, sTM, or TFPI levels. In contrast, TF ⁺-MP activity was significantly elevated in the DIC group versus the non-DIC group (p < 0.001). TF⁺ -MP activity strongly correlated with the ISTH DIC score (r² = 0.9; p = 0.003), SOFA score (r² = 0.8; p = 0.001), D-dimer (p = 0.001), FDP (p = 0.01), and sTM (p = 0.02), but not with TFPI levels. Thrombin generation parameters differed markedly: the DIC group had a significantly longer lag-time and reduced mean rate index (MRI) and Peak of thrombin generation (p = 0.001). Lag time was positively correlated with DIC and SOFA scores, while endogenous thrombin potential (ETP) and peak values were negatively correlated. Inflammatory markers (IL-1β , CRP, TNF-α and PCT) were significantly elevated in the DIC group (p < 0.01). TF⁺ -MP activity correlated significantly with IL-1 β, TNF-α , CRP, and PCT levels. TF⁺ -MP activity was significantly higher in non-survivors compared to survivors at 20 days (p < 0.005), while total MP concentrations did not differ. Conclusion: The ROADMAP-Sepsis study highlights TF⁺ -MP activity as a central mediator of thromboinflammation and a key contributor to the development of DIC, endothelial dysfunction, and increased sepsis severity. Unlike total microparticle levels, TF ⁺-MP activity showed strong correlations with inflammatory markers, markers of hypercoagulability, and 20-day mortality. These findings support the clinical relevance of TF⁺-MP activity as a prognostic biomarker for identifying septic patients at high risk of deterioration. Ongoing studies from our group are focused on validating its incorporation into predictive scoring systems to guide risk stratification and therapeutic decision-making.

Introduction: Cancer cells (CaCe) expressing Tissue Factor (TF) initiate thrombin generation and fibrin formation, contributing to cancer-associated hypercoagulability. The resulting fibrin clot shield (FCS) functions both as a protective barrier against therapeutic agents and as a scaffold facilitating CaCe migration. Our objective was to isolate and characterize cancer cells that migrate into the FCS and to determine involvement of a selective process. Materials and Methods: Highly procoagulant pancreatic CaCe (BxPC3) and invasive, highly procoagulant breast CaCe (MDA-MB-231) were cultured in RPMI-1640 medium supplemented with 10% human platelet-poor plasma (PPP) to induce FCS formation (Chi Mai Thromb Res 2024). Mechanical disruption of the clot enabled the isolation of CaCe embedded within the FCS, hereafter referred to as “clot cancer cells” (clot-CaCe). These clot-CaCe, as well as the remaining “paternal CaCe” (CaCe remaining adherent to the wells after clot removal), were subsequently cultured separately in RPMI-1640. BXPC3 and MDA-MB-231 CaCe cultured in conventional RPMI-1640 medium without plasma or coagulation activation to serve as the “control” condition. Cell viability and proliferation were assessed using the crystal violet assay. The procoagulant activity of the cancer cells was evaluated using the calibrated automated thrombogram (CAT® from Diagnostica Stago), as previously described (Tran et al Thromb Res 2024). Scanning electron microscopy (SEM) was used to analyze FCS structure and CaCe invasion within the fibrin network (method described in Tran et al Thromb Res 2024). Results: Clot-CaCe embedded in the fibrin network were successfully isolated and subsequently cultured. BxPC3 clot-CaCe showed higher proliferative capacity than their paternal adherent counterparts. Both BxPC3 and MDA-MB-231 clot-CaCe retained thrombin-generating capacity comparable to that of the parental CaCe. Moreover, both clot- and parental BxPC3 cells and MDA-MB-231 cells induced fibrin network formation when cultured in the presence of PPP. When clot-CaCe or parental-CaCe were exposed to normal PPP, they induced thrombin generation to a similar extent, with no significant difference compared to control CaCe. The SEM analysis showed that the clot-CaCe migrated into the FCS similarly to the paternal and control CaCe populations. However, fibrin networks generated by clot-CaCe exhibited a looser architecture, characterized by thicker fibers, larger pores, and fewer intersections compared to those formed by paternal adherent cells or control CaCe. Conclusion: We developed a novel methodology to study cancer cell behavior within fibrin networks formed through their inherent procoagulant activity. This study provides, for the first time, evidence that CaCe embedded within the fibrin clot network remain viable and preserve their proliferative and procoagulant potential to a similar degree as their progenitors. Moreover, clot-CaCe are capable of generating new fibrin networks. The resulting fibrin appears structurally softer. This observation supports the hypothesis that clot-embedded cancer cells may represent a distinct clone derived from the parental CaCe population. The newly formed fibrin network, in turn, serves as a scaffold supporting further CaCe migration and the formation of new colonies. Taken together, the data presented herein suggest that the fibrin clot network may promote selective survival and clonal expansion of cancer cells, pointing to a potentially novel mechanism of clonal selection and preservation within the hostile tumor microenvironment. The fibrin clot shield may serve as a protective barrier, shielding cancer cells from immune surveillance and the cytotoxic effects of targeted or cell-based anticancer therapies. Overall, this model provides a valuable tool to investigate cancer-associated hypercoagulability, thrombosis, disease recurrence, and therapeutic resistance. References: Tran et al. The procoagulant signature of cancer cells drives fibrin network formation in the tumor microenvironment and impacts its quality: Implications in cancer cell migration and the resistance to anticancer agents. Thromb Res. 2024;238:172–183. doi:10.1016/j.thromres.2024.04.015

Introduction: Neoangogenesis, vascular mimicry and highly permeable blood vessels expose cancer cells to coagulation factors from plasma. The expression of Tissue Factor (TF) by cancer cells leads to local hypercoagubility in the tumor microenvironment. We aimed to study the ability of cancer cells to induce fibrin formation in the tumor microenvironment, and the role of fibrin in cancer cells' physiology. Materials and methods: Highly procoagulant pancreatic cancer cells (BXPC3), highly procoagulant and invasive breast cancer cells (MDA-MB231), weakly procoagulant and non-invasive breast cancer cells (MCF7) were cultured in the presence of of normal human platelet-poor plasma (PPP) diluted 10% in RPMI-1640 media (PPP-media). Structural characteristics of fibrin clot were analyzed using Scanning Electron Microscopy. Fibrinolytic activities were studied by quantifying tissue-type plasminogen activator (t-PA) and D-Dimer concentrations. Cancer cells' invasion was observed using Laser Scanning Confocal Microscopy. The efficacity of paclitaxel (PTX) and 4-hydroxytamoxifen (4OHTam), at their IC50 concentrations, on cancer cells viability in the absence and presence of fibrin clot shields was assessed. Results: Culture of cancer cells in the presence of PPP-media led to the formation of Fibrin Clot Shields (FCS) in a “bird's nest-like” architecture. MDA-MB231 cells led to the formation of the most dense FCS with finest fibers, smallest pores and highest number of intersections, as contrast to MCF7 cells. The structure of FCS formed by BXPC3 are intermediated. Adding 1 pM of TF to the culture media of MCF7 led to FCS with similar structures to those of BXPC3 cells. MCF7 cells do not possess fibrinolytic activity as contrast to the other two cell lines. All of the three cancer cell lines showed invasion ability in the presence of FCS. The presence of FCS also abrogated the efficacity of PTX and 4OHTam in inducing cell death. Conclusions: The procoagulant fingerprint of cancer cells leads to the formation of FCS in the microenvironment. We showed for the first time a clear image of the architecture of FCS formed by cancer cells. FCS serve as scaffolds for cell invasion and protection against anticancer treatments such as PTX and 4OHTam. These results suggested FCS as a new mechanism for resistance to treatments and a potential new target to improve treatments' efficacity.

Introduction: Cancer cells induce hypercoagulability in the tumoral microenvironment by expressing Tissue Factor (TF). We aimed to study the impact of the procoagulant signature of cancer cells on the quality and structure of fibrin network. We also studied the impact of fibrin clot shield (FCS) on the efficiency of anticancer agents and the migration of cancer cells. Materials and methods: Pancreatic cancer cells BXPC3 and breast cancer cells MDA-MB231 and MCF7, were cultured in the presence of normal Platelet Poor Plasma (PPP), diluted 10 % in conditioning media. Their potential to induce thrombin generation and their fibrinolytic activity were assessed. The structure of fibrin network was analyzed with Scanning Electron Microscopy (SEM). Cancer cells ' mobility with fibrin clot and their interactions with fibrin were observed. Cancer cells were treated with paclitaxel (PTX) or 4-hydroxy-tamoxifen (4OHTam) in the presence or absence of FCS. Results: Cancer cells, in presence of PPP, induced fibrin network formation. High TF-expressing cancer cells (BXPC3 and MDA-MB23 cells), led to dense fibrin network with fine fibers. Low TF expressing cells MCF7 led to thick fibers. Exogenous TF enhanced the density of fibrin network formed by MCF7 cells. Cancer cells through their inherent profibrinolytic potential migrated within the fiber scaffold. The BXPC3 and MCF7 cells moved in clusters whereas the MDA-MB231 cells moved individually within the fibrin network. FCS decreased the efficiency of PTX and 4OHTam on the viability of cancer cells. Conclusions: The procoagulant signature of cancer cells is determinant for the quality and structure of fibrin network in the microenvironment. Original SEM images show the architecture of "bird 's nest " -like fibrin network being in touch with the cell membranes and surrounding cancer cells. Fibrin network constructed by triggering thrombin generation by cancer cells, provides a scaffold for cell migration. Fibrin clot shields protect cancer cells against PTX and 4OHTam.

Introduction: Sickle cell disease (SCD) is linked to hypercoagulability and risk of venous thromboembolism. It is characterised by high concentrations of erythrocyte-derived microparticles (EC-d-MP). In addition, in SCD endothelial cells and monocytes can be activated, which in turn are sources of tissue factor bearing MPs (TF-MP). Their impact on the thrombin generation process remains unclear. Aim: We hypothesised that in SCD TF-MP and procoagulant phospholipids from endothelial cells and monocytes are implicated in blood hypercoagulability and potentially enhance thrombin generation . Materials and methods: Consecutive patients with steady state SCD (n=68) and 31 age and sex-matched patients in painful crisis (n=31) and 30 healthy individuals (controls) were included. Flow cytometric analysis was performed with a precise number of standard beads (Megamix®) to determine MVs count, and specific conjugated antibodies were used to determine the cellular origin of the MVs. Microparticles derived from erythrocytes (RBd-MP), platelets (Pltd-MP), monocyte (Mod-MP), endothelial cell (ECd-MP) were identified using an anti-CD235a, CD61/CD41a, CD14, CB106 and CD41 monoclonal antibodies and FITC labelled annexin V. Total TF-MP was evaluated by CY-Quant MV-TF activity (BioCytex ,Marseille, France). Cellular origin of TF on MPs, samples were triple labelled with Cy5-labeled annexin V, a cell type-specific PE-labeled MoAb against either CD14 or CD144 and a FITC- MoAb against TF. Thrombin generation (TG) in platelet poor plasma was measured by CAT assay using PPP-reagent 5pM in presence and absence of thrombomodulin (TM) . Procoagulant phospholipid dependent activity was assessed by the Procoag-PPL assay, TM and TFPI were measured by Asserachrom TM and Asserachrom TFPI (Stago, Asnieres, France). Results: The mean age of SCD patients in painful crisis and steady state were 25.1 ± 9.6 (range: 18-39 years) and 26.3 ± 8.1 years (range: 18-40 years), respectively. The mean age in the control group was 27.4 ± 7.2 years (range: 16-36 years). Total MPs were significantly elevated for both groups of patients with SCD in steady as compared to the controls (p<0.001). A significant difference was observed between the steady state patients and crisis patients (p<0.001). In both groups of SCD patients, RBd-MP, Pltd-MP expressing or not PS, ECd-MP and Mod-MP were significantly increased as compared to the control group (p<0.001). The RBd-MP, RBd-MP-PS+, Ed-MP and Mod-MP were significantly elevated in patients in crisis compared to those at steady state SCD (p<0.001, p<0.001, p<0.05, p<0.001 respectively). The TF-MPs were derived from endothelial cells and monocytes but not from red blood cells or platelets. Patients either in crisis or at steady state SCD showed significantly higher total TF-MPs T as compared to the control group (p<0.001). Patients in crisis had significantly higher TF-MP as compared to those at steady state SCD (p<0.001). Mod-MP-TF+ and Ed-MP-TF+ were significantly elevated in both patient groups as compared to the control group (p<0.001). Patients in crisis had significantly higher MP levels as compared to those at steady state SCD (p<0.001). TG was significantly increased in SCD patients as compared to the control. Among thrombogram parameters, the mean rate index (MRI) and Peak of thrombin were significantly higher in the SCD-patient group as compared to the control group (p<0.01). The MRI was significantly increased in patients with crisis as compared to those at steady state SCD (p<0.01). The lag-time and the ETP were not significantly different between SCD patients and the control group. The ETP +/- TM ratio increased significantly in both patients' groups as compared to the controls (p<0.001), with a significant difference between steady state and crisis (p<0.01). The TM, and TFPI and the PPL-ct were significantly decreased in patients as compared to the controls (p<0.01). The PPL-ct was inversely correlated with the levels of Rb-MP/PS+ and Pd-MP/PS+.( p<0.02).The Rb-MP/PS+ were positively correlated with MRI (p<0.02). The concentration of the total MPs-TF+, Mod-MP-TF+ was positively correlated with MRI and Peak (p<0.01). Conclusion: In SCD patients the TF bearing MPs derived from endothelial cells or monocytes could, contribute to thrombotic events and may be involved in painful crisis. Circulating MVs TF + may be considered as a potential biomarker for disease severity in SCD patients.

Background The link between blood hypercoagulability, endothelial cell activation and infertility or in vitro fertilization (IVF) failure is a puzzling issue. Activation of platelets, endothelial cells and blood coagulation, proinflammatory and angiogenetic potential play important role in implantation and early embryonic development. Biomarkers of cellular hypercoagulability are associated with high risk of IVF failure. Soluble Leukemia Inhibitory Factor (LIF), an interleukin 6 class cytokine expressed in the trophectoderm of the developing embryo is an important regulator in the establishment of pregnancy. Endoglin (Eng), a co-receptor for TGF-β1 and TGF-β3 is implicated in vascular complications of pregnancy and particularly in preeclampsia. Aim: The prospective longitudinal monocentric observational cohort study ROADMAP-IVF, evaluated the synergistic prognostic value of LIF and Eng with the biomarkers of cellular hypercoagulability in predicting the endometrial receptivity of fresh IVF cycles. Materials and Methods. The ROADMAP-IVF enrolled 40 women eligible for IVF with normal blood count, PT, aPTT, Fg, renal and liver function. The control group (CG) consisted of 30 healthy women with history of uncomplicated pregnancies. Exclusion criteria: Use of anticoagulant or antiplatelet agents during the last 30 days before inclusion. Known cardiovascular disease, active cancer. Active corticosteroid treatment. Primary end-point: Echographically documented pregnancy at 7 weeks from implantation. Blood was collected on day 2 (D2) from the natural cycle and 6 days after hormone treatment initiation corresponding to D8 of the cycle. Procoagulant phospholipid-dependent clotting time (PPL-ct), tissue factor (TF), thrombomodulin (TM), von Willebrand factor (vWF) and D-Dimers (DDi) were measured with assays from Diagnostica Stago (Asnieres, France). P- and E-Selectin, LIF and Eng were measured with ELISA. Thrombin generation (TG) with the TF 5 pM PPP-Reagent ® was assessed on Calibrated Automated Thrombogram (Stago, France). The cut-off values for studied biomarkers have been selected on the basis of ROC analysis. Univariate and multivariate logistic regression analysis examined the associations between the biomarkers and the study outcome. Results: 40 women were enrolled in the study. In 11 women pregnancy was confirmed with ultrasound and in 23 women no pregnancy was echographically documented. Age was not significantly different between the IVF (41 ys; range 22 - 48 ys) and the control group (40 ys; range 20 - 46 ys). At D2 and D8 the DDi and LIF were significantly higher in IVFG as compared to the CG. At D2 most of the patients had PPL-ct, DDi and MRI of TG higher than the upper normal limit (UNL). At D8 only TF was significantly increased as compared to the CG. Analytical data are presented in Table 1. Age did not correlate with the levels of the studied biomarkers. Significant correlations (p<0.05) were found between DDi and ETP (r=0.35); TM, TF and P-Selectin (r=0.4) at D2; TF, LIF and Eng (r=0.5) at D2 and D8; P-Selectin and PPL-ct (r=-0.4) at D2; P-Selectin and TM (r=0.4), E-Selectin (r=0.5), LIF (0.4) at D2; E-Selectin and P-Selectin (r=0.5), TF (r=0.3), and LIF (r=0.4) at D2. The ROC analysis showed that IVF failure was associate with DDi increase at D2 (AUC = 0.64); P-Selectin increase at D2(AUC = 0.57) and D8 (AUC = 0.61); TM increase at D8 (AUC = 0.61); TF increase at D8 (AUC = 0.57); LIF increase at D8 (AUC = 0.59); ETP increase at D2 (AUC = 0.67) and D8 (AUC = 0.67); Peak increase at D2 (AUC = 0.64) and D8 (AUC = 0.61). Prolongation of PPL-ct on D8 was predictive for positive pregnancy outcome(AUC 0.64). Conclusion The ROADMAP-IVF study showed that at least one out of four women eligible for IVF present biological evidence of activation of platelets, endothelial cells, blood coagulation or LIF upregulation. Hormone treatment administration does not significantly alter the proinflammatory or the hypercoagulable state. Among the studied biomarkers the levels of LIF, DDi, P-Selectine, TM and TF as well as the status of thrombin generation and PPL-ct showed a significant predictive value for the IVF outcome, particularly when measured at 6 days after hormone treatment administration. These findings will lead to the elaboration of an risk assessment model for IVF failure combining the selected biomarkers of hypercoagulability with LIF.

Backround: Thromboembolism occurs in about 10% of patients with multiple myeloma (MM). The incidence of VTE is higher in newly diagnosed MM patients as compared to those with relapsed or refractory disease and it is higher during the first 3 to 6 months after the initial diagnosis and treatment initiation. Some of the treatments administered to patients with MM are independent risk factors for VTE. Immunomodulatory agents (IMiDs) among anti-myeloma treatments stand out as having a considerable prothrombotic effect. Recognizing the significant risk associated with the use of immunomodulatory agents (the International Myeloma Working Group (IMWG) 2014 statement, and the European Myeloma Network Guidelines in 2015 both included guidance on the prevention of VTE in MM patients who receive IMiDs. The prospective, longitudinal observational study ROADMAP-MM CAT was designed to explore alternative strategies for the development of risk stratification tools in patients with multiple myeloma. To this target we evaluated the baseline profile of hypercoagulability in multiple myeloma patients with various disease status. Blood borne hypercoagulabity partly consisted of enhanced thrombin generation, a common phenomenon in patients with malignancies . It remains to be seen whether this phenomenon appears for multiple myeloma patients during the physical course of the disease. Aim: We conducted a study to explore the relationship between stages of MM and alterations of various thrombosis-related biomarkers in patients with MM and their relationship with MM therapy. Materials and Methods Patients with MM (n=162) were recruited and stratified to the following groups: 59 newly diagnosed treatment-naïve patients (ND), 49 patients receiving IMiDs (IM), 52 in complete remission (CR) and 12 patients in partial remission on IMiDs (PR/IM). Patients on anticoagulant treatment were excluded from the study. The control group (CG) consisted of 30 healthy age and sex-matched individuals. Samples of platelet-poor plasma (PPP) were assessed for thrombin generation (TG) with the TF 5pM PPP-Reagent® on Calibrated Automated Thrombogram (Diagnostica Stago, France). Plasminogen activator inhibitor-1 (PAI-1), soluble endothelial protein C receptor (sEPCR), and soluble vascular cell adhesion molecule-1 (sVCAM-1), Procoag-PPL were measured using antibody-based ELISA kits (Invitrogen International Inc., CA, USA, and Diagnostica Stago, France ). TF expression MPC-derived microparticles (MPC-dMPs) were detected using a Zymuphen MP-TF activity kit (Hyphen BioMed, Neuville sur Oise, France). The upper and lower normal limits (LNL and UNL) were calculated by the mean±2 SD. Results A total of 162 patients were enrolled (age 66.0±12.0 yrs; 53% male). Distribution of disease stage was as follows: 32% ISS I, 23% ISS II, 45% ISS III. Bone disease was present in 71% of patients and 19% of patients had high risk cytogenetic lesions. Patients with ongoing MM (ND, IM, PR/IM) had significantly lower Peak, ETP and MRI as compared to the CG. In contrast, patients in CR had Peak, ETP, MRI values similar to the CG. Patients with PR had lower ETP and MRI values as compared to the CR group. In ND 9% had TG >UNL and 22% had TGUNL and 67% had TGUNL and 35% had TGUNL and 12% had TG

Background: Hypercoagulability is a common blood alteration in newly diagnosed chemotherapy naïve patients with multiple myeloma. Multiple myeloma (MM) figures among malignancies that significantly increase the risk of venous thromboembolism (VTE). The rate of VTE is higher at the time of diagnosis and during the first months following initiation of first line therapy; approximately 10% of newly diagnosed MM (NDMM) will develop a VTE. Despite adequate thromboprophylaxis as per guidelines, the risk of residual VTE is not eliminated and remains as high as 12%. The optimization of VTE prevention in patients with MM is an unmet need. The exact mechanism of the increased risk of VTE is not yet fully understood. The identification of the procoagulant potential of cancer cells, which is related principally to tissue factor (TF) expression, attracts particular interest. Aim: We conducted a longitudinal prospective observational study, to explore the relationship of MM with cellular and plasma hypercoagulability as well as tissue factor positive microparticles, aiming to identify the most relevant biomarkers, which could be used in a Risk Assessment Model (RAM) for VTE in combination with clinical risk factors. Methods: Newly diagnosed patients (182) with multiple myeloma (NDMM) were prospectively enrolled. Patients were followed up for 12 months and the primary end-point was symptomatic objectively confirmed VTE. Patients were risk stratified and received thromboprophylaxis (none, aspirin on low molecular weight heparin (LMWH) based on previously published recommendations by the IMWG and EMN. Prior to treatment initiation and thromboprophylaxis initiation baseline biomarkers were obtained: Thrombin generation (TGT) in citrated PPP was assessed with the Thrombogram-Thrombinoscope® assay using PPP-reagent® 5 pm TF by Diagnostica Stago. The levels of P-Selectin and heparanase in plasma were measured with ELISA Kit (Cusabio Biotech and R&D Systems respectively). The procoagulant phospholipids clotting time was measured with STA-Procoag-PPL®, Levels of Factor VIIa were measured by Staclot VIIa-rTF®, D-Dimers (DDi) by Liatest D-Di (Diagnostica Stago, France), and Tissue Factor activity (TFa) by specific clotting based home test. TF expression MPC-derived microparticles (MPC-dMPs) were detected using a Zymuphen MP-TF activity kit (Hyphen BioMed, Neuville sur Oise, France). These were compared against values in a population of healthy individuals (n=30). Results: The distribution of patients enrolled in the study is as follows: Median age was 67 years (36-86) and 52% of the population was male. Median time to follow up was 7 months (1-12 months). The control group (30 healthy individuals). The overall rate of symptomatic VTE during follow-up was 11.5% (n = 21 patients). Eleven out of 21 events (52%) occurred within 3 months from treatment initiation. Six of these patients did not receive any thromboprophylaxis; six patients were on aspirin at the time of the event and three were on LMWH.At inclusion, patients showed significantly increased levels of TFa, FVIIa, D-Dimers and FM, and significantly shorter Procoag-PPL® as compared to the group of healthy individuals. Levels of P-selectin and TM were significantly lower in patients as compared to healthy individuals. The levels of heparanase were not significantly different in the group of patients as compared to the healthy individuals. Overall thrombin generation was attenuated in patients compared to healthy individuals. Lag-time and ttPeak were significantly increased and Peak, MRI, and ETP were significantly lower as compared to the control group, MP-TF activity measured in NDMM significantly increased 1.46± 0.38 pg/mL compared to control subjects 0.19 ± 0.05 pg/mL (Table 1). Multivariate logistic regression analysis demonstrated that ETP, Procoag-PPL® and MP-TF were independently associated with VTE occurrence. (Table 2) Conclusion: The prospective ROADMAP-CAT-MM study demonstrates the presence of pronounced cellular hypercoagulability in newly diagnosed chemotherapy naïve patients with MM characterized by decreased Procoag-PPL® clotting time, enhanced endothelial cell activation, and exhausted thrombin generation. The Procoag-PPL clotting time and the ETP and MP-TF were found to be independently associated with the risk of VTE and can be prospectively incorporated into a RAM for VTE in MM.

Background The ROADMAP-EOP study aimed to identify clinically relevant biomarkers of hypercoagulability for the identification of pregnant women at risk of early onset preeclampsia worsening. Methods The ROADMAP-EOP observational single center retrospective case–control study was conducted in Greece (Centre for Human Reproduction, Genesis Athens Clinic, Athens, Greece) from July 2020 to July and enrolled pregnant women diagnosed with EOP stratified in mild EOP group (n = 34) and severe EOP group (n = 15) as well as women with uncomplicated pregnancy (control group; n = 35). All women were assessed with thromboelastometry (ROTEM®), Calibrated Automated Thrombogram®, tissue factor activity (TFa), procoagulant phospholipid dependentclotting time (Procoag-PPL®), Proteins S (PS), TFPI, D-dimer, antithrombin (AT), thrombomodulin (TM), fibrinogen, prothrombin time (PT) and activated partial thromboplastin time (aPTT). The primary study end-point was severe earlyonset preeclampsia. Principal component analysis (PCA) was performed. Results The PCA analysis showed that a score composed of the lag-time, ttPeak and Procoag-PPL accurately predicted severe EOP (sensitivity 71.4%, specificity 61.8%, and AUC of the ROC analysis 0.953). Conclusion The pilot ROADMAP-EOP shows that activation of endothelial cells and blood hypercoagulability are driven events in the worsening of EOP. Among a large panel of biomarkers and coagulation assays, thrombingeneration test and procoagulant phospholipid dependent clotting time emerged as clinically relevant for the evaluation of the risk of severe EOP. This methodology for the development of a new clinic-biological risk assessment model for prompt identification of pregnant women at risk of severe EOP must be validated in a large multi-centerprospective study.

Background: Patients with COVID 19 disease have haemostatic dysfunction and are at higher risk of thrombotic complications. Although age and sex are a major risk factor for outcome. Even though the incidence of COVID 19 seems to be similar in both genders, men are at a higher risk of a worse outcome, with the odds up to 2.8 times higher than those for women. It is not known whether sex hormones contribute to this preposition. The real impact of sex hormone levels on the severity of COVID 19 remains to be demonstrated. Objectives: We aimed to investigate the impact of variations in sex hormone levels on the hypercoagulability and severity of COVID 19 in women and men according to age. Methods: 186 patients with symptomatic newly diagnosed Covid 19 were enrolled in the study. Specially we included postmenaupausal women (>50 years) and age matched men. Participants were divided into two groups: moderate Covid-19 (ARDS) and severe Covid-19. The control group consisted of 60 healthy individuals, with the same age as patients. Biomarkers of hypercoagulability and endothelial cell activation: Procoagulant phospholipid (PPL) TFPI, D-Dimer, Soluble thrombomodulin , Tissue factor activity, Thrombin generation (TG) with the Calibrated Automated Thrombogram (CAT) were all measured with assays were from Diagnostica Stago, Asnières, France. Total testosterone, estradiol, and progesterone were measured by ELISA method (Abcam, France). IL-6, Complement C3a and C5a by Human IL-6 ELISA kit, ThermoFisher (Scientific Asnieres-sur-Seine, France). The Upper Normal Limit (UNL) and the Lower Normal Limit (LNL) for each studied biomarker were defined in the control group as follows: UNL= mean + 2 standard deviation (SD), and LNL: = mean - 2 SD. Results: The levels of biomarkers of hypercoagulability, endothelial cell activation and inflammation in patients and controls are reported in Table 1. Thrombogram was marked by significantly prolonged lag time, prolonged time to peak and decreased ETP compared to the control group. The comparison between females and males showed that the lag-time was over the UNL in 18% of men over 50 years versus 8% in men under 50 years, and in 10% and 0% of women respectively. ETP values were below the LNL in 82% of men over 50 years versus 63% for men under 50 years and in 54% and 92% for women respectively. In the same way, the peak of thrombin was over the UNL in 39% and 15% for men over 50 and men under 50 versus 12% and 0% for women respectively. The TFPI were over the UNL in 99% of men over 50 years and in 84% of men under 50 years versus 91% and 87% for women respectively. D-Di increased in 88% of patients under or over 50 years independently of the group. The PPL were shorter than the LNL in 90 % and 80% of men over and under 50 years respectively, and for women in 86% and 77% respectively. The levels of TM were significantly lower in women for the 2 groups in comparison with men over or under 50 years. The levels of C3a, were above the UNL in 86%, and 63% for men over and under 50 years respectively and in 55% and 87% of women over and under 50 years respectively. The testosterone levels in males both younger and older than 50 years were significatively below the lower limit of the normal range of each age group. The estradiol levels were in normal range for women but significantly increased men (Table2). Stratification by the severity of COVID-19 showed that the testosterone levels were significantly lower in men who developed ARDS or severe COVID-19 than in those who did not (p>0.05). In men, the estradiol levels were significantly increased but the difference was not significant between those with severe or mild COVID-19. Males who developed severe COVID-19 had a lower progesterone level. No statistically significant difference in testosterone or progesterone levels were found in the women compared to normal range, the estradiol level was lower in females with ARDS (Table 3.)Relationship analysis of sex hormones with markers of immune activation, testosterone levels and estradiol/testosterone were correlated with, IL-6, fibrinogen, and D-Di. Conclusion: Our study highlights that disturbance in circulating sex hormone levels is a hallmark of critical COVID-19 in males and reinforces the concept that determination of testosterone and E2/T ratio in association with biomarkers of hypercoagulability upon diagnosis might lead to an a accurate risk assessment tool for prompt identification of patients at high risk of COVID-19 worsening. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background. Hypercoagulable state and endothelial cell activation are common alterations in patients with COVID-19. Nevertheless, the hypothesis of persistent hypercoagulability and endothelial cell activation following recovery from COVID-19 remains an unresolved issue. Objectives. To investigate the persistence of endothelial cell activation and hypercoagulability after recovery from COVID-19. Patients/Methods. COVID-19 survivors (n = 208) and 30 healthy individuals were enrolled in this study. The following biomarkers were measured: procoagulant phospholipid-dependent clotting time (PPL-ct), D-Dimer, fibrin monomers (FM), free Tissue factor pathway inhibitor (free-TFP)I, heparinase, and soluble thrombomodulin (sTM). Antibodies against SARS-CoV-2 (IgG and IgA) were also measured. Results. The median interval between symptom onset and screening for SARS-CoV-2 antibodies was 62 days (IQR = 22 days). Survivors showed significantly higher levels of D-Dimers, FM, TFPI, and heparanase as compared to that of the control group. Survivors had significantly shorter PPL-ct. Elevated D-dimer was associated with older age. Elevated FM was associated with female gender. Elevated heparanase was independently associated with male gender. Decreased Procoag-PPL clotting time was associated with female gender. One out of four of COVID-19 survivors showed increase at least one biomarker of endothelial cell activation or hypercoagulability. Conclusions. Two months after onset of COVID-19, a significant activation of endothelial cells and in vivo thrombin generation persists in at least one out of four survivors of COVID-19. The clinical relevance of these biomarkers in the diagnosis and follow-up of patients with long COVID-19 merits to be evaluated in a prospective clinical study.

Bakground: Preeclampsia occurs in up to 7% of pregnancies in Western Countries and is a leading cause of maternal and fetal morbidity and mortality. Classification of preeclampsia is based on the timing of symptoms' onset and the severity of the clinical status. Accordingly, early and late preeclampsia are characterized by symptom onset before or after 32-34 weeks of gestation respectively. Pathogenesis of preeclampsia is characterized by abnormal placental vascular development with impaired invasion of the uterine spiral arteries by trophoblasts, resulting in fetal complications and maternal endothelial dysfunction. Intact antithrombotic properties of the endothelial cells and efficient regulation of thrombin generation and platelet activation at the microenvironment of the uterus are necessary conditions for successful trophoblast invasion and development of feto-maternal circulation during normal pregnancy. , Prompt identification of pregnant women at risk of preeclampsia worsening could help to anticipated therapeutic intervention and improvement of the clinical outcome. Aim: The ROADMAP-Early Onset Preeclampsia study (ROADMAP-EOP) aiming to respond to this unmet need assessed the clinical relevance of biomarkers of hypercoagulability and endothelial activation for prompt identification of pregnant women at high risk of preeclampsia. Materials and Methods: An observational single center retrospective case-control study was conducted in pregnant women diagnosed with preeclampsia from July 2020 to July 2021. Pregnant women were retrospectively enrolled upon EOP diagnosis and stratified in mild EOP group (n=34) and severe EOP group (n=15 according to the criteria of the International Society for the Study of Hypertension in Pregnancy ). Eligible women were included in the study before initiation of any treatment for preeclampsia. The control group (n=35) consisted of women with uncomplicated pregnancy. All women were assessed with thromboelastometry, Calibrated Automated Thrombo-gram®, tissue factor activity (TFa), Procoagulant phospholipid dependent clotting time (Procoag-PPL), Proteins S, TFPI, D-dimer, antithrombin, thrombomodulin, fibrinogen prothrombin time and activated partial thromboplastin time. Primary and secondary end points were severe EOP and EOP respectively. Principal component analysis (PCA) was performed to check for associations among the biomarkers and the clinical out-come. Results: There were no significant differences regarding age, gravidity, gestational age and the body mass index (BMI) between EOP groups and normal pregnancy as well as within the EOP groups. Platelet counts were similar in healthy pregnant women, and mild EOP group, and significantly lower in severe EOP (p<0.05). No other statistically significant differences were found between-groups. The PCA showed that the repartition of data allowed to discriminate the patients with EOP from healthy pregnant women as well as patients with mild EPO from those with severe EOP. When biomarkers of hypercoagulability were considered according to the severity of EOP in PCA analysis the lag-time and PPL clotting time were independently associated with the risk of severe EOP (Table 1and Figure 1)). The increase of the lag-time by 1 min was associated with a 20.2-fold increase of the risk of severe EOP. The marginal effects curve shows that for a lag-time longer than 2.6 min, the probability of severe EOP was higher than 50% .Longer Procoag-PPL clotting time was associated with lower risk of severe EOP. The marginal effects curve, showed that for Procoag-PPL clotting time longer than 58.75 sec the probability of severe EOP decreased by 50%. These data suggest that a biological score composed of the Procoag-PPL® clotting time and thrombin generation (Throbmogram-Thrombinoscope® PPP-reagent 1 pM-TF as-say) could be a useful tool to identify pregnant women with high probability of EOP Conclusion: the Procoag-PPL® clotting time and thrombin generation assay reflect cellular derived hypercoagulability and allow accurate screening of pregnant women for the prompt identification of those at risk of EOP and the evaluation of its severity. This concept has to be validated in a large prospective cohort trial. . An external validation of the proposed methodology needs to be performed in a large prospective observational study.

In some patients, SARS-CoV-2 infection induces cytokine storm, hypercoagulability and endothelial cell activation leading to worsening of COVID-19, intubation and death. Prompt identification of patients at risk of intubation is an urgent need. Objectives. To derive a prognostic score for the risk of intubation or death in patients with COVID-19 admitted in intensive care unit (ICU), by assessing biomarkers of hypercoagulability, endothelial cell activation and inflammation and a large panel of clinical analytes. Design, Setting and Participants. A prospective, observational study enrolled 118 patients with COVID-19 admitted in the ICU. On the first day of ICU admission, all patients were assessed for biomarkers (protein C, protein S, antithrombin, D-Dimer, fibrin monomers, FVIIa, FV, FXII, FXII, FVIII, FvW antigen, fibrinogen, procoagulant phospholipid dependent clotting time, TFPI, thrombomodulin, P-selectin, heparinase, microparticles exposing TF, IL-6, complement C3a, C5a, thrombin generation, PT, aPTT, hemogram, platelet count) and clinical predictors. Main Outcomes and Measures. The clinical outcomes were intubation and mortality during hospitalization in ICU. Results: The intubation and mortality rates were 70% and 18%, respectively. The COMPASS-COVID-19-ICU score composed of P-Selectin, D-Dimer, free TFPI, TF activity, IL-6 and FXII, age and duration of hospitalization predicted the risk of intubation or death with high sensitivity and specificity (0.90 and 0.92, respectively). Conclusions and Relevance. COVID-19 is related to severe endothelial cell activation and hypercoagulability orchestrated in the context of inflammation. The COMPASS-COVID-19-ICU risk assessment model is accurate for the evaluation of the risk of mechanical ventilation and death in patients with critical COVID-19. The COMPASS-COVID-19-ICU score is feasible in tertiary hospitals and could be placed in the diagnostic procedure of personalized medical management and prompt therapeutic intervention.

Background: COVID-19 has been associated with hypercoagulability, endothelial cell injury and frequent thrombotic complications resulting both from direct effects of the virus on the endothelium and from the 'cytokine storm' resulting from the host's immune response. Since the COVID-19 vaccines have been shown to effectively prevent symptomatic infection including hospital admissions and severe disease, the risk of COVID-19-related thrombosis should be expected to (almost) disappear in vaccinated individuals. However, some rare cases of venous thrombosis have been reported in individuals vaccinated with mRNA vaccines. Thus, there is a sharp contrast between the clinical or experimental data reported in the literature on COVID-19 and on the rare thrombotic events observed after the vaccination with these vaccines. This phenomenon raised some scepticism of even some fear about the safety of these vaccines which could compromise the adhesion of the citizens in the vaccination program. Aims: We conducted a prospective observational study, to explore the impact of vaccination with the BNT162b2 (Pfizer/BioNTech) on blood hypercoagulability and endothelial cell activation and to investigate if this is modified by the presence of active cancer. Methods: In total 229 subjects were prospectively included in the study from April to June 2021. Subjects were stratified in three predefined groups: 127 vaccinated patients with active cancer (VOnco group), 72 vaccinated health care workers (VHcw group) and 30 non vaccinated health individuals (Control group). Blood samples were obtained 2 days after the administration of the first dose of BNT162b2 vaccine and collected in Vacutainer® tubes (0.109 mol/L trisodium citrate). Platelet poor plasma (PPP) was prepared by double centrifugation at 2000 g for 20 minutes at room temperature and plasma aliquots were stored at -80°C until assayed. Samples of PPP were assessed for thrombin generation (TG) with PPP-Reagent® (Thrombogram-Thrombinoscope assay with PPP-Reagent®TF 5pM), E-selectin, D-dimers, (D-Di), Tissue Factor (TFa), procoagulant phospholipid-dependent clotting time (Procag-PPL) and von Willebrand factor (vWF), thrombomodulin (TM), tissue factor pathway inhibitor (TFPI), and platelet factor 4 (PF4). All assays were from Diagnostica Stago (France). The upper and lower normal limits (UNL and LNL) for each biomarker were calculated by the mean±2SD for the control group. Results: All vaccinated subjects showed significantly increased levels of PF4 (71% >UNL, p<0.001), D-Dimers (74% >UNL, p<0.01), vWF (60% >UNL, p<0.01), FVIII (62% >UNL, p<0.01) and shorter Procoag-PPL clotting time (96% UNL, p<0.01), ETP (38% >UNL, p<0.01) and MRI (66% >UNL, p<0.01) but no differences in lag-time in vaccinated subjects as compared to the control group. Vaccinated subjects did not show any increase at the levels of TFa, TFPI, TM and E-selectin in comparison with the control group. The studied biomarkers were not significantly different between the VOnco and VHcw groups. Conclusion: The ROADMAP-COVID-19-Vaccine study shows that administration of the first dose of the BNT162b2 vaccine induced significant platelet activation documented by shorter Procoag-PPL associated with increased levels of PF4. Plasma hypercoagulability was less frequent in vaccinated individuals whereas there was no evidence of significant endothelial cells activation after vaccination. Interestingly, the presence of active cancer was not associated with an enhancement of platelet activation, hypercoagulability, or endothelial cell activation after the vaccination. Probably, the generated antibodies against the spike protein or lead to platelet activation in a FcyRIIa dependent manner that results in PF4 release. The implication of the mild inflammatory reaction triggered by the vaccination could be another possible pathway leading to platelet activation. Nevertheless, vaccination does not provoke endothelial activation even in patients with cancer. The findings of the ROADMAP-COVID-19-Vaccine study support the concept administration of mRNA based vaccines does not directly cause a systematic hypercoagulability. Disclosures Gligorov: Roche-Genentech: Research Funding; Novartis: Research Funding; Onxeo: Research Funding; Daichi: Research Funding; MSD: Research Funding; Eisai: Research Funding; Genomic Heatlh: Research Funding; Ipsen: Research Funding; Macrogenics: Research Funding; Pfizer: Research Funding. Terpos: Novartis: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Genesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; GSK: Honoraria, Research Funding. Dimopoulos: Amgen: Honoraria; BMS: Honoraria; Janssen: Honoraria; Beigene: Honoraria; Takeda: Honoraria.

Introduction:Different coagulations abnormalities have been referred in women with early oncet preeclampsia (EOP), but there are only few studies comparing those changes regarding to the severity of the disease. Aim:In this study we aimed to investigate the differences between the coagulation profile in women with mild and severe preeclampsia. Methods:This is an observational retrospective case-control study. Plasma samples were collected from 84 women divided into three groups, the healthy pregnant (HP) group (n=35), the mild preeclampsia (MP) group (n=34) and the severe preeclampsia (SP) group (n=15). The study population general characteristics are shown in Table 1. We studied the following biomarkers of hypercoagulability and endothelial cell activation: Tissue factor activity (TFa), Procoagulant phospholipid activity (PPL), Protein S, D-Dimers, Antithrombin, thrombomodulin, TFPI levels. All women were assessed with classic coagulation tests (aPTT and PT) fibrinogen levels and hemogram. Statistical analysis was performed using the PASW Statistics 17.0.2 (SPSS Inc.) for Windows. Results:Women with preeclampsia - mild or severe- showed significant increase of TFPI, TFa and TMa levels as compared to healthy pregnant women. No significant difference of TFPI, TFa was observed between MP and SP groups. In contrast, TMa levels were significantly increased in SP as compared to MP group. The ratio TFa/TFPI was also lower in SP as compared to MP-group. Women in MP or SP group had similarly shorter PPL clotting time as compared to HP group. D-dimer levels were increased in women with preeclampsia as compared to the HP group. D-Dimer levels were significantly higher in SP as compared to MP group. The levels of free PS activity in HP as well as MP and SP groups were lower than normal range in non-pregnant women and the value in MP was significantly lower than that of the HP or SP. Fibrinogen levels were not significantly different in the three studied groups of pregnant women. Prothrombin time was found to be increased in cases as compared to that in the controls. The mean value of prothrombin time in mild preeclampsia was 13.24±0.80 seconds and in severe preeclampsia it was seconds 14.77±0.96 and in pregnant controls 12.23±0.59 seconds (p<0.05 and p<0.001 respectively). The mean prothrombin time was found to increase with increasing severity of disease (p<0.001). The mean activated partial thromboplastin time were increased in mild preeclampsia and was 32.64±1.83 seconds and in severe preeclampsia it was 35.59±1.53 seconds and in pregnant controls 29.53±1.62 seconds (p<0.001). The activated partial thromboplastin time was found to increase with increasing severity of disease (p<0.001). The antithrombin III decreased in severe SP and MP or compared to pregnant controls (76.33±4.32 and 88.06±9.68 versus 95.40±0.36 respectively; p<0.001). This decrease is more pronounced in SP compared to MP (p<0.001). Conclusions:Preeclampsia is associated with endothelial cell activation as documented by the increase of TFa, soluble TM levels and TFPI levels in plasma. Release of soluble thrombomoduline and TFPI rather than TFa by endothelial cells appear to be related with degree of preeclampsia severity. Women with preeclampsia showed marked decrease of PPL clotting time indicating enhanced platelet activation that was independent of the severity of preeclampsia. In contrast, women with severe preeclampsia showed signs of enhanced hypercoagulability documented by the increase of D-dimer levels consumption of natural coagulation inhibitors and particularly of AT. This phenomenon tended to be reflected on the prolongation of PT and aPTT in women with severe preeclampsia. Disclosures No relevant conflicts of interest to declare.

Introduction: Preeclampsia is a frequent vascular complication of pregnancy and figures among the major causes of maternal and neonatal morbidity and mortality. Early diagnosis and prompt, targeted treatment remain a unmet need. Hypercoagulability and endothelial cell activation are among the principal pathogenetic mechanisms in patients with preeclempsia. Development of diagnostic algorithms including clinically relevant biomarkers of hypercoagulability is expect to improve the management of preeclampsia. Among the numerous coagulation test, Global Coagulation Assays (GCA) such as thrombogram and thromboelastometry, could be of potential value for the evaluation of blood hypercoagulability. They provide information, on thrombin generation process, clot formation kinetics, clot firmness and even fibrinolysis potential. Aim: In this study we investigated the clinical accuracy of whole blood thromboelastometry (ROTEM®), and thrombin generation assay (calibrated automated thrombography: CAT® assay) to identify women with preeclampsia and we tried to compare their sensitivity. Methods: An observational retrospective case-control study was conducted. Plasma samples were collected from 84 women divided into three groups, the healthy pregnant (HP) group (n=35), the mild preeclampsia (MP) group (n=34) and the severe preeclampsia (SP) group (n=15). Thromboelastometry in whole blood was performed on ROTEM delta instrument (Tem Innovations GmbH, Werfen, Munich, Germany) with INTEM reagent. Thrombin generation triggered by PPP reagent low® (1 pM TF and 4µM phospholipid) was measured in platelet poor plasma. Thrombogram was also assessed in the presence or absence of thrombomodulin and the corresponding ration was calculated. Blood was collected at the diagnosis of preeclampsia (groups MP and SP) or at the equivalent months of pregnancy in the control group (HP). Statistical analysis was performed using the PASW Statistics 17.0.2 (SPSS Inc.) for Windows. Results: Thromboelastometry analysis showed that the clotting time (CT) was significantly longer in SP group as compared to MP and HP group. Both preeclampsia groups had longer clot formation time (CFT as compared to HP-group. MP-group had longer CFT as compared to SP-group. The α angle was significantly lower in SP-group as compared to the HP and MP groups. The maximum clot firmness was significantly higher in MP groups as compared to either HP or SP-group. The mean lysis (ML) was lower in both preeclampsia groups as compared to the HP group (Table 1). Thrombogram analysis showed that the lag-time of thrombin generation was significantly longer in both MP and SP groups as compared to HP group. Moreover, SP group showed significantly longer lag -time as compared to MP-group. Peak and the endogenous thrombin potential (ETP) were significantly higher in MP group as compared to either HP or SP groups. The mean rate index of the propagation phase of thrombin generation was not significantly different among the three groups whereas the thrombomodulin ratio for the ETP was significantly shorter in the SP-group (Table 2). Both tests showed a significant prolongation of the initiation phase of blood coagulation (reflected on CT and lag-time) in SP. The levels of clotting factors and fibrinogen were normal in all patients and none was on anticoagulant treatment. Thus, this prolongation reflects changes at the levels of TFPI and Thrmbomodulin reflecting an endothelial cell activation. ROTEM showed a decrease of the α-angle and MCF in SP group which is related with a lower platelet count in these patients. ROTEM showed enhanced fibrinolysis in both MP and SP groups Women with MP showed higher Peak and ETP than SP, MP showed higher ratio of ETP (TM+/TM-) than SP. Conclusion: The two GCA proved complementary information on the status of blood coagulation in pregnant women with preeclampsia. ROTEM provides information on clot formation kinetics and clot firmness as well as on fibrinolysis activation, which allow to differentiate SP from HP. However, the capacity of the assay for identification of patients with MP is limited. Thrombin generation assay showed a distinct profile between the three groups, which allowed differentiating the MP from HP as well as from SP. Disclosures No relevant conflicts of interest to declare.